Previous studies have demonstrated that a peripheral corticotropin releasing factor (PCRF), which possesses CRF-like immunological and biological activity, is released from injured tissue during inflammation. Additional studies have demonstrated that synthetic CRF possesses potent antinociceptive and anti-inflammatory activity when injected directly into inflamed tissue. However, there is a gap in knowledge regarding the identity and regulation of release of peripheral CRF and regarding the mechanism(s) of action for the antinociceptive and anti-inflammatory effects of synthetic CRF. The objectives of this proposal will 1) identify the peripheral origin of CRF released during inflammation; 2) characterize the regulation of peripheral CRF release; 3) determine the effects of CRF on modulating the peripheral release of markers of primary afferents (immunoreactive substance P, CGRP), sympathetic fibers (immunoreactive NPY) and leukocytes (immunoreactive LTB4 and beta-endorphin); and 4) determine whether CRF-induced modulation of primary afferents, sympathetics or leukocytes contributes to its mechanism of action. Samples of PCRF and inflammatory mediators will be collected by subcutaneous placement of microdialysis probes into carrageenan-inflamed hindpaws of rats. The PCRF will be compared to synthetic CRF for its physicochemical similarity (HPLC elution profile, protease and oxidation susceptibility) and quantitated for CRF-like immunoreactivity (using a CRF radioimmunoassay) and CRF-like biological activity (using release of immunoreactive beta-endorphin from pituitary cultures). Levels of immunoreactive substance P, CGRP, NPY, LTB4 and beta-endorphin will be measured by previously validated radioimmunoassays. Collectively, this research proposal will evaluate the hypothesis of a new peripheral neuroendocrine axis, in which the process of inflammation leads to the local release and actions of an antinociceptive/anti-inflammatory peptide, CRF.